Vascularized skin flap and preparation method thereof

Abstract

The invention provides a vascularized skin flap and a preparation method thereof, and belongs to the field of tissue engineering materials, and the preparation method of the vascularized skin flap comprises the step of providing muscular tissue and artificial blood vessels. The muscular tissue is cooled, the cooled muscular tissue is subjected to trepanning treatment, and the first structure is obtained. And performing contraction treatment on the artificial blood vessel, and implanting the contracted artificial blood vessel into the opening of the first structure to obtain a second structure. And performing expansion treatment on the artificial blood vessel in the second structure to obtain a third structure. And performing decellularization treatment on the third structure to obtain the vascularized skin flap. Wherein the outer diameter of the artificial blood vessel is larger than the inner diameter of the opening. According to the technical scheme, the connection stability of the artificial blood vessel and muscular tissue can be improved, the ingrowth of new blood vessels and the vascularization process of vascularized flaps can be further promoted, the survival rate of flap transplantation is increased, and therefore the vascularization effect of the vascularized flaps is improved.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, specifically to a vascularized flap and its preparation method. Background Technology

[0002] Skin flap transplantation is an important method for repairing large-area skin defects, soft tissue injuries, and organ reconstruction. Traditional skin flap transplantation relies on the patient's own donor site, which suffers from problems such as donor site damage, limited tissue volume, and low morphological matching. The emergence of tissue-engineered flaps has provided a new approach to repair materials; however, in practical applications, they still face the core challenge of insufficient vascularization. Due to the lack of a functional vascular network, implanted flaps often experience ischemic necrosis due to insufficient blood supply to the central area, leading to transplantation failure.

[0003] In related technologies, the connection stability between artificial blood vessels and muscle tissue is not good. They are mostly fixed by simple physical interlocking or suturing, which easily leads to problems such as displacement, detachment, or poor adhesion between the artificial blood vessels and muscle tissue. Summary of the Invention

[0004] In view of the technical problems existing in the background art, this application proposes a vascularized flap and its preparation method. The preparation method of the vascularized flap can improve the connection stability between artificial blood vessels and muscle tissue, further promote the ingrowth of new blood vessels and the vascularization process of the vascularized flap, improve the survival rate of flap transplantation, thereby improving the vascularization effect of the vascularized flap and reducing the risk of ischemic necrosis after transplantation.

[0005] To achieve the above objectives, in a first aspect, embodiments of this application provide a method for preparing a vascularized flap, comprising: Provide muscle tissue and artificial blood vessels; The muscle tissue is cooled, and the cooled muscle tissue is then perforated to obtain the first structure. The artificial blood vessel is shrunk and then implanted into the opening of the first structure to obtain the second structure. The artificial blood vessel in the second structure is expanded to obtain the third structure; The third structure is decellularized to obtain a vascularized flap; among which, The outer diameter of the artificial blood vessel is larger than the inner diameter of the opening.

[0006] Furthermore, the cooling temperature is -20℃ to 4℃; and / or, The cooling process takes 2-24 hours.

[0007] Furthermore, shrinkage treatment includes cooling treatment, and expansion treatment includes heating treatment.

[0008] Furthermore, the cooling treatment temperature is 0℃-5℃; and / or, The cooling process takes 1-4 hours; and / or, The heating treatment temperature is 25℃-37℃; and / or, The heating process takes 1-6 hours.

[0009] Furthermore, the shrinkage treatment includes axial stretching treatment, and the expansion treatment includes releasing the axial stretching; and / or, The shrinkage process includes negative pressure compression, and the expansion process includes releasing the negative pressure compression.

[0010] Furthermore, the porosity of the vascularized flap is 65%-90%; and / or, In the opening process, multiple openings are set, with the center distance between adjacent openings being 2cm-5cm; and / or, In the opening process, multiple openings are provided, and the extension directions of the multiple openings are arranged in parallel; and / or, Decellularization treatment includes physical shaking treatment and chemical washing treatment.

[0011] Furthermore, the outer diameter of the artificial blood vessel is 0.2 mm to 1.5 mm larger than the inner diameter of the opening; and / or, The inner diameter of the artificial blood vessel is 0.5mm-3mm; and / or, Artificial blood vessels include biodegradable artificial blood vessels made of at least one biodegradable material selected from polyglycolic acid, polylactic acid-glycolic acid copolymer, polylactic acid, polydioxanone, or polycaprolactone; and / or, The inner wall of the artificial blood vessel is coated with a bioactive coating, which comprises at least one of gelatin, chitosan, collagen, or vascular endothelial growth factor; and / or, Muscle tissue includes at least one of the muscle tissues of pigs, cattle, sheep, and rabbits.

[0012] Furthermore, the artificial blood vessel includes interconnected connecting segments and anastomotic segments, with the connecting segments disposed within the opening and the anastomotic segments extending to the outside of the muscle tissue.

[0013] Furthermore, the thickness of the muscle tissue is 3mm-15mm; and / or, The length of the anastomotic segment is 2mm-10mm; and / or, The length of the artificial blood vessel is greater than the length of the opening, so as to form two anastomosing segments outside the muscle tissue.

[0014] Secondly, the present invention also proposes a vascularized flap, which is prepared by the above-described method for preparing a vascularized flap.

[0015] The beneficial effects of this invention are: In the technical solution of this invention, cooling treatment reduces the elasticity and toughness of muscle tissue, making the tissue texture denser and more stable. This reduces tissue deformation and rebound during the opening process, ensuring the dimensional accuracy and morphological integrity of the opening. The opening treatment creates a hole in the muscle tissue, providing installation space for the artificial blood vessel. The contraction treatment allows the outer diameter of the artificial blood vessel to be smoothly implanted into the opening in the muscle tissue in a contracted state. Since the original outer diameter of the artificial blood vessel is larger than the inner diameter of the opening, during the expansion treatment after implantation, the artificial blood vessel will return to its original size, thus forming a tight interference fit with the inner wall of the opening. Decellularization treatment removes cellular components and immunogenic substances from the muscle tissue, preserving its extracellular matrix scaffold structure. The interference fit enhances the connection strength between the artificial blood vessel and the muscle tissue, reducing problems such as displacement, detachment, or poor adhesion that easily occur in traditional physical embedding or suturing methods.

[0016] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of this application, the accompanying drawings used in this application will be briefly described below. Obviously, the drawings described below are merely some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without any creative effort.

[0018] Figure 1 This invention provides an embodiment of a vascularized flap; Figure 2 yes Figure 1 A cross-sectional schematic diagram of a vascularized flap.

[0019] Explanation of reference numerals in the attached figures: 100. Vascularized flap; 1. Muscle tissue; 11. Opening; 2. Artificial blood vessel; 21. Connecting segment; 22. Anastomosis segment; 3. Bioactive coating. Detailed Implementation

[0020] The embodiments of the technical solution of this application will now be described in detail with reference to the accompanying drawings. These embodiments are only used to more clearly illustrate the technical solution of this application and are therefore merely examples, and should not be used to limit the scope of protection of this application.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms “comprising” and “having”, and any variations thereof, in the specification, claims, and foregoing description of the drawings are intended to cover non-exclusive inclusion.

[0022] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0023] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0024] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0026] In the description of the embodiments of this application, the technical terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.

[0027] In the description of the embodiments of this application, unless otherwise expressly specified and limited, technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. For those skilled in the art, the specific meaning of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0028] Skin flap transplantation is an important method for repairing large-area skin defects, soft tissue injuries, and organ reconstruction. Traditional skin flap transplantation relies on the patient's own donor site, which suffers from problems such as donor site damage, limited tissue volume, and low morphological matching. The emergence of tissue-engineered flaps has provided a new approach to repair materials; however, in practical applications, they still face the core challenge of insufficient vascularization. Due to the lack of a functional vascular network, implanted flaps often experience ischemic necrosis due to insufficient blood supply to the central area, leading to transplantation failure.

[0029] In related technologies, the connection stability between the artificial blood vessel 2 and the muscle tissue 1 is not good. It is often fixed by simple physical interlocking or suturing, which easily leads to problems such as displacement, detachment, or poor adhesion between the artificial blood vessel 2 and the muscle tissue 1.

[0030] To address the aforementioned technical problems, in a first aspect, the present invention proposes a method for preparing a vascularized flap 100, comprising: Provide muscle tissue 1 and artificial blood vessel 2; The muscle tissue 1 is cooled, and the cooled muscle tissue 1 is then perforated to obtain the first structure. The artificial blood vessel 2 is shrunk, and the shrunk artificial blood vessel 2 is implanted into the opening 11 of the first structure to obtain the second structure. The artificial blood vessel 2 in the second structure is expanded to obtain the third structure; The third structure was decellularized to obtain a 100-cell vascularized flap; among which, The outer diameter of the artificial blood vessel 2 is larger than the inner diameter of the opening 11.

[0031] like Figure 1As shown, in the technical solution of this invention, cooling treatment reduces the elasticity and toughness of muscle tissue 1, making the tissue texture denser and more stable. This reduces tissue deformation and rebound during the opening process 11, ensuring the dimensional accuracy and morphological integrity of the opening 11. The opening treatment creates an opening 11 in the muscle tissue 1, providing installation space for the artificial blood vessel 2. The contraction treatment allows the outer diameter of the artificial blood vessel 2 to be smoothly implanted into the opening 11 of the muscle tissue 1 in a contracted state. Since the original outer diameter of the artificial blood vessel 2 is larger than the inner diameter of the opening 11, during the expansion treatment after implantation, the artificial blood vessel 2 will return to its original size, thus forming a tight interference fit with the inner wall of the opening 11. Decellularization treatment removes cellular components and immunogenic substances from the muscle tissue 1, preserving its extracellular matrix scaffold structure. The interference fit enhances the connection strength between the artificial blood vessel 2 and the muscle tissue 1, reducing problems such as displacement, detachment, or poor adhesion of the artificial blood vessel 2 that are prone to occur in traditional physical interlocking or suturing methods. In addition, the interference fit interface not only achieves mechanical interlocking and enhances structural integrity, but also forms a localized densification area around the artificial blood vessel 2, improving the mechanical support of the matrix, and at the same time facilitating the ingrowth of host cells and the integration of the vascular network.

[0032] It can be explained that the perforation process refers to the use of specific tools (such as puncture needles, micro drills, etc.) to make holes in the muscle tissue 1 according to the preset position, number, spacing and direction, so as to form a channel for the implantation of artificial blood vessels 2.

[0033] In some embodiments, the cooling temperature is -20°C to 4°C.

[0034] In this embodiment, by limiting the temperature of the cooling process to this range, the muscle tissue 1 can be hardened while reducing the risk of frostbite to the tissue cell structure or denaturation of proteins due to excessively low temperature.

[0035] In some embodiments, the cooling process takes 2-24 hours.

[0036] In this embodiment, by limiting the cooling time to this range, the muscle tissue 1 can be made to reach the required hardness.

[0037] Understandably, shrinkage can be achieved in a variety of ways.

[0038] In some embodiments, the shrinkage process includes a cooling process, and the expansion process includes a heating process.

[0039] In this embodiment, the size of the artificial blood vessel 2 is controlled by temperature changes. This method is simple and easy to control, enabling precise regulation of the contraction and expansion processes of the artificial blood vessel 2. Furthermore, it is worth mentioning that this embodiment also uses temperature control, which works synergistically with cooling treatment to reduce the complexity of temperature control in the overall preparation process, thereby improving operational convenience and stability.

[0040] In some embodiments, the temperature for the cooling process is 0°C-5°C.

[0041] In this embodiment, by limiting the temperature of the cooling treatment to this range, the material of the artificial blood vessel 2 can physically shrink due to the temperature reduction, achieving the size that facilitates implantation of the opening 11, while avoiding the adverse effects of excessively low temperature on the biocompatibility or degradation performance of the artificial blood vessel 2.

[0042] In some embodiments, the cooling process takes 1-4 hours.

[0043] In this embodiment, by limiting the cooling time to this range, the artificial blood vessel 2 can fully contract under the set low temperature conditions to obtain a stable contraction size and ensure the smooth progress of the implantation process.

[0044] In some embodiments, the temperature for the heating process is 25°C-37°C.

[0045] In this embodiment, by limiting the temperature of the heating process to this range, the contracted artificial blood vessel 2 can be restored to its original outer diameter, thereby forming a tight interference fit with the inner wall of the opening 11, while reducing the risk of damage to the material properties of the artificial blood vessel 2 or thermal damage to the muscle tissue 1 caused by excessively high temperature.

[0046] In some embodiments, the heating process takes 1-6 hours.

[0047] In this embodiment, by limiting the heating time to this range, the artificial blood vessel 2 can have enough time to complete the expansion process, so that its outer diameter can be fully restored and form a stable interference fit with the inner wall of the opening 11.

[0048] In some embodiments, the shrinkage process includes an axial stretching process, and the expansion process includes releasing the axial stretching.

[0049] In this embodiment, an axial tensile force is applied to the artificial blood vessel 2, causing it to elastically deform along its length, thereby reducing its outer diameter and facilitating implantation into the opening 11. When the axial tension is released, the artificial blood vessel 2 rebounds to its original size under its own elastic restoring force, achieving an interference fit with the inner wall of the opening 11. This mechanical contraction and expansion has the characteristics of rapid response and high dimensional recovery accuracy, and is suitable for artificial blood vessel 2 materials that are sensitive to temperature or where temperature control is inconvenient.

[0050] In some embodiments, the shrinkage process includes a negative pressure compression process, and the expansion process includes releasing the negative pressure compression.

[0051] In this embodiment, by placing the artificial blood vessel 2 in a sealed container and applying a negative pressure environment, the vessel wall undergoes radial contraction under the influence of the internal and external pressure difference, thereby reducing its outer diameter to facilitate passage through the opening. When the negative pressure is released, the artificial blood vessel 2 returns to its initial state and fits tightly against the inner wall of the opening 11. This method allows for precise control of the negative pressure value to quantify the degree of contraction and is applicable to artificial blood vessels 2 made of various materials.

[0052] In some embodiments, the porosity of the vascularized flap 100 is 65%-90%.

[0053] In this embodiment, by controlling the porosity within this range, it is beneficial for the penetration of nutrients and oxygen, providing a good microenvironment for subsequent cell growth and tissue regeneration, while also ensuring that the vascularized flap 100 has a certain mechanical strength.

[0054] In some embodiments, during the opening process, multiple openings 11 are provided, and the center distance between adjacent openings 11 is 2cm-5cm.

[0055] In this embodiment, by limiting the center distance between adjacent openings 11 to this range, artificial blood vessels 2 can be reasonably distributed within the limited space of muscle tissue 1, so that any tissue site within the vascularized flap 100 is within the nutrient diffusion radius, reducing the problem of central ischemic necrosis caused by insufficient blood supply.

[0056] In some embodiments, during the opening process, multiple openings 11 are provided, and the extension directions of the multiple openings 11 are arranged in parallel.

[0057] In this embodiment, the parallel arrangement facilitates the positioning and implantation of the artificial blood vessel 2 during the surgical procedure. It also facilitates the subsequent directional docking and integration of the host vascular network and the artificial blood vessel 2. At the same time, it can form a uniformly distributed blood flow channel inside the vascularized flap 100, thereby improving the blood perfusion efficiency and nutritional supply balance of the vascularized flap 100.

[0058] In some embodiments, the decellularization process includes physical shaking and chemical washing.

[0059] In this embodiment, the synergistic effect of physical vibration and chemical washing can remove cellular components and immunogenic substances from muscle tissue, while preserving the natural structure and bioactive components of the extracellular matrix to the greatest extent possible, providing an ideal scaffold environment for subsequent attachment, migration and angiogenesis of host cells.

[0060] In some embodiments, the outer diameter of the artificial blood vessel 2 is 0.2 mm to 1.5 mm larger than the inner diameter of the opening 11.

[0061] In this embodiment, by limiting the difference between the outer diameter of the artificial blood vessel and the inner diameter of the opening to this range, a stable interference fit is formed after expansion, which can reduce the insufficient connection strength caused by too small interference and the excessive compression damage to muscle tissue caused by too large interference.

[0062] In some embodiments, the inner diameter of the artificial blood vessel 2 is 0.5mm-3mm.

[0063] In this embodiment, by limiting the inner diameter of the artificial blood vessel 2 to this range, the size characteristics of human microvessels can be better simulated, which is conducive to the smooth flow of blood in the artificial blood vessel 2, reduces blood flow resistance, and also provides a suitable spatial environment for the attachment, migration and proliferation of vascular endothelial cells, promotes the endothelialization process of the artificial blood vessel 2, and thus improves the biocompatibility and long-term patency of the vascularized flap 100.

[0064] In some embodiments, the artificial blood vessel 2 comprises a biodegradable artificial blood vessel 2 made of at least one biodegradable material selected from polyglycolic acid, polylactic acid-glycolic acid copolymer, polylactic acid, polydioxanone or polycaprolactone.

[0065] In this embodiment, the artificial blood vessel 2 is prepared by a biodegradable material, so that after the artificial blood vessel 2 has completed its phased mission of supporting and guiding blood vessel regeneration, it can be gradually degraded in the body and absorbed by the body, avoiding complications such as foreign body reaction, chronic inflammation or vascular stenosis that may be caused by the long-term retention of traditional non-degradable blood vessel materials in the body.

[0066] Specifically, polyglycolic acid (PGA) exhibits good biocompatibility and a relatively fast degradation rate, providing stable mechanical support for angiogenesis in the early stages. The degradation rate of polylactic acid-glycolic acid copolymer (PLA-GA) can be controlled by adjusting the ratio of the two monomers, better matching the angiogenesis cycle. PLA has high mechanical strength and a relatively long degradation cycle, making it suitable for scenarios requiring long-term support. Polydioxanone (PDA) possesses excellent flexibility and tensile strength, maintaining good structural integrity during in vivo degradation. Polycaprolactone (PCL) degrades slowly, which is beneficial for long-term vascular structural stability and functional reconstruction. The metabolic products generated during the degradation of these biodegradable materials are water and carbon dioxide, which can be excreted through normal physiological pathways without adverse effects on the body, thus improving the safety and applicability of vascularized flap 100 in clinical applications.

[0067] In some embodiments, the inner wall of the artificial blood vessel 2 is provided with a bioactive coating 3, which comprises at least one of gelatin, chitosan, collagen or vascular endothelial growth factor.

[0068] In this embodiment, the bioactive coating 3 can enhance its biocompatibility and angiogenesis-promoting ability.

[0069] Specifically, gelatin and collagen, as natural biomolecules, possess excellent hydrophilicity and cell adhesion, providing a suitable matrix environment for the adhesion and proliferation of vascular endothelial cells. Chitosan not only has antibacterial properties, reducing the risk of early infection after implantation, but also promotes platelet adhesion and aggregation, aiding in coagulation and initial vascular closure. Vascular endothelial growth factor (VEGF), as an important cytokine, specifically acts on vascular endothelial cells, promoting their proliferation, migration, and lumen formation, accelerating the endothelialization process of the artificial blood vessel 2, thereby improving the long-term patency rate of the artificial blood vessel 2 and reducing the occurrence of complications such as thrombosis. These bioactive components work synergistically, enabling the artificial blood vessel 2 to integrate with the host tissue more quickly after implantation and guiding the regeneration and reconstruction of its own vascular network.

[0070] In some embodiments, muscle tissue 1 includes at least one of the muscle tissues of pigs, cattle, sheep, and rabbits.

[0071] In this embodiment, muscle tissue 1 from common mammals such as pigs, cattle, sheep and rabbits is used as raw material, which has the advantages of wide availability and relatively low cost. At the same time, the muscle tissue 1 of these animals has certain similarities with human muscle tissue 1 in terms of tissue structure, extracellular matrix composition and mechanical properties. After decellularization, the natural three-dimensional scaffold structure and bioactive components can be well preserved, providing a suitable microenvironment for subsequent ingrowth of host cells and tissue regeneration.

[0072] Specifically, porcine muscle tissue 1 has a fibrous arrangement structure and collagen content that are relatively similar to human muscle tissue 1, and its acquisition cost is low and its supply is stable; bovine muscle tissue 1 is tough and rich in extracellular matrix, and after decellularization, it can form a scaffold structure with high mechanical strength, which is suitable for flap applications with high support performance requirements; sheep muscle tissue 1 has relatively low immunogenicity, and after decellularization, it has a low risk of inducing host immune rejection, which is beneficial to improving the biocompatibility of vascularized flap 100; rabbit muscle tissue 1 is small in size and easy to perform standardized experimental operations and processing.

[0073] like Figure 2 As shown, in some embodiments, the artificial blood vessel 2 includes a connecting segment 21 and an anastomosis segment 22 connected to each other, the connecting segment 21 being disposed within the opening 11, and the anastomosis segment 22 extending to the outside of the muscle tissue 1.

[0074] In this embodiment, the connecting segment 21 serves as the core part of the implantation opening 11, with its outer diameter forming an interference fit with the inner diameter of the opening 11, ensuring the fixation stability of the artificial blood vessel 2 within the muscle tissue 1 and reducing displacement issues. Meanwhile, the anastomosis segment 22 extends to the outside of the muscle tissue 1, providing ample operating space and clear connection points for anastomosis operations with the host's own blood vessels (such as arteries and veins) during the surgical procedure. This simplifies the clinical surgical process, reduces the technical difficulty of vascular anastomosis, and facilitates surgical suturing and anastomosis.

[0075] In some embodiments, the thickness of muscle tissue 1 is 3mm-15mm.

[0076] In this embodiment, by limiting the thickness of the muscle tissue 1 to this range, sufficient space can be provided for the implantation and distribution of the artificial blood vessel 2 to meet the needs of the vascularized flap 100 for the construction of the blood supply network.

[0077] In some embodiments, the length of the anastomosis segment 22 is 2mm-10mm.

[0078] In this embodiment, by limiting the length of the anastomosis segment 22 to this range, a suitable operational margin can be provided for the precise docking of the artificial blood vessel 2 and the host blood vessel in actual use, which helps to improve the success rate of the anastomosis surgery.

[0079] In some embodiments, the length of the artificial blood vessel 2 is greater than the length of the opening 11, so as to form two anastomotic segments 22 outside the muscle tissue 1.

[0080] In this embodiment, the two anastomosing segments 22 facilitate bidirectional anastomosis with the host blood vessels (e.g., one end connects to an artery and the other end connects to a vein). In addition, a through-flow blood channel can be constructed inside the vascularized flap 100 to ensure that blood can flow in from one end and out from the other end, forming a complete circulatory path and improving the blood perfusion efficiency and nutrient exchange capacity of the vascularized flap 100.

[0081] Secondly, the present invention also proposes a vascularized flap 100, which is prepared by the above-described method for preparing a vascularized flap 100. This vascularized flap 100 possesses all the beneficial effects of the above-described method for preparing a vascularized flap 100, which will not be elaborated further here.

[0082] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0083] I. Preparation Method Example 1 S1. Provide fresh pig muscle tissue 1 (8mm thick) and artificial blood vessel 2: Artificial blood vessel 2 is woven from polyglycolic acid (PGA) filaments (1.0mm inner diameter, 1.5mm outer diameter), and its inner wall is coated with a gelatin coating containing vascular endothelial growth factor (VEGF).

[0084] S2. The muscle tissue 1 was frozen at -10°C for 6 hours to harden it. Then, using a precision drill with an inner diameter of 1.2 mm, three parallel holes 11 were drilled in the muscle tissue, with a center-to-center distance of 3 cm between adjacent holes 11, to obtain the first structure.

[0085] S3. Place the artificial blood vessel 2 in a 2°C environment for 2 hours to cool it down, causing its outer diameter to shrink to approximately 1.3 mm. Carefully insert the shrunken artificial blood vessel into the opening 11 of the first structure to obtain the second structure.

[0086] S4. The second structure is heated in an environment of 32°C for 2 hours to allow the artificial blood vessel 2 to expand and return to its original outer diameter (1.5 mm), thereby forming an interference fit with the inner wall of the opening 11 (1.2 mm) to obtain the third structure.

[0087] S5. The third structure was placed in PBS buffer (phosphate-buffered saline) containing 1% Triton X-100 (octylphenyl polyoxyethylene ether, a nonionic surfactant) and 0.1% sodium dodecyl sulfate (SDS), and treated on a shaker at 100 rpm for 48 hours. During the treatment, the PBS buffer containing 1% Triton X-100 and 0.1% SDS was changed every 16 hours, for a total of three times. Afterward, the flap was continuously washed with a large amount of fresh PBS buffer for 72 hours to complete the decellularization process, obtaining the vascularized flap of Example 1.

[0088] Example 2 The difference from Example 1 is as follows: muscle tissue 1 is bovine muscle (10 mm thick), and the cooling treatment temperature is -15°C for 12 hours. Artificial blood vessel 2 is made of polylactic acid-glycolic acid copolymer (PLGA, LA:GA = 90:10), with an inner diameter of 2.0 mm and an outer diameter of 2.8 mm. The opening 11 has an inner diameter of 2.5 mm. The artificial blood vessel 2 is cooled at 5°C for 3 hours and then heated at 37°C for 4 hours. The remaining steps are similar to those in Example 1 and will not be elaborated further, resulting in the vascularized flap of Example 2.

[0089] Example 3 The difference from Example 1 is that the artificial blood vessel 2 is contracted using an axial stretching method. The artificial blood vessel 2 (outer diameter 1.5 mm) is stretched 10% axially, causing it to contract radially to approximately 1.3 mm outer diameter before being implanted into the opening 11 (inner diameter 1.2 mm). After implantation, the stretching is released, allowing it to expand radially back to its original shape, forming an interference fit. The remaining steps are similar to those in Example 1 and will not be elaborated further, resulting in the vascularized flap of Example 3.

[0090] Example 4 The difference from Example 1 is that six openings 11 are provided, arranged in two parallel rows, with a center-to-center distance of 2 cm between adjacent openings. The difference between the outer diameter of the artificial blood vessel 2 and the inner diameter of the opening 11 is 0.8 mm. The remaining steps are similar to those in Example 1 and will not be described in detail here, resulting in the vascularized flap of Example 4.

[0091] Example 5 The difference from Example 1 is that the muscle tissue 1 is rabbit muscle (5 mm thick), the artificial blood vessel 2 is made of polycaprolactone (PCL) material with an inner diameter of 0.5 mm and an outer diameter of 0.9 mm. The inner diameter of the opening 11 is 0.7 mm. The remaining steps are similar to those in Example 1 and will not be described in detail here, resulting in the vascularized flap of Example 5.

[0092] Comparative Example 1 The difference from Example 1 is that the muscle tissue was not cooled; instead, the soft muscle tissue was opened directly at room temperature. The remaining steps were similar to those in Example 1 and will not be described in detail here, resulting in the vascularized flap of Comparative Example 1.

[0093] Comparative Example 2 The difference from Example 1 is that no expansion treatment was performed after implanting the artificial blood vessel. The remaining steps are similar to those in Example 1 and will not be described in detail here, resulting in the vascularized flap of Comparative Example 2.

[0094] II. Testing Methods 1. Interface shear strength test Testing instrument: Universal testing machine (Model: Instron5943, maximum load: 5kN).

[0095] Sample preparation: From the vascularized flaps prepared in each embodiment and comparative example, a rectangular sample (length × width: 15mm × 10mm) containing a single artificial blood vessel and its surrounding muscle tissue matrix was cut, ensuring that the artificial blood vessel was located in the center of the sample.

[0096] Test method: A custom-designed dual-clamp system was used, with the upper clamp securing both ends of the artificial blood vessel and the lower clamp securing the muscle tissue matrix. Shear force was applied along the axial direction of the blood vessel at a constant speed until the artificial blood vessel separated from the matrix interface.

[0097] Test conditions: Temperature: 37±0.5℃ (simulating physiological environment, tested in constant temperature PBS solution) Stretching speed: 1mm / min Humidity: >90%RH (tested in a solution environment, isolated from air) Data processing: Record the maximum shear force (Fmax), and calculate the interfacial shear strength (τ) using the following formula: τ=Fmax / A Where A is the theoretical contact area between the artificial blood vessel and the matrix (calculated based on the outer diameter of the blood vessel and the length of the opening).

[0098] 2. Overall tear resistance test of the skin flap Testing instrument: Universal testing machine (Model: Instron5943, maximum load: 5kN).

[0099] Sample preparation: Cut the complete skin flap into a standard dumbbell-shaped specimen (gauge length 20mm, width 5mm), ensuring that the specimen contains at least one artificial blood vessel.

[0100] Test method: Apply a unidirectional tensile force along the long axis of the specimen until the specimen breaks.

[0101] Test conditions: Temperature: 37±0.5℃ (tested in PBS solution) Stretching speed: 5mm / min Humidity: >90%RH Data processing: Record the maximum tensile force (Fmax) and the fracture location, and calculate the tensile strength.

[0102] III. Analysis of Test Results for Each Embodiment and Comparative Example Table 1 Mechanical properties of Examples 1-5 and Comparative Examples 1-3 Results Analysis and Summary: As can be seen from the test results in Table 1, the vascularized flaps prepared in Examples 1-5 of this application are superior to those in Comparative Examples 1-2 in terms of both interfacial shear strength and overall tear resistance.

[0103] Example 1 uses a combination of cryo-curing followed by drilling and thermal expansion interference fit. The interfacial shear strength reaches 58.2 kPa and the overall tear resistance is 1.25 MPa, indicating that the process can effectively achieve a tight bond between the artificial blood vessel and the muscle tissue matrix.

[0104] Example 2 uses bovine muscle tissue and adjusts the temperature treatment parameters. The interfacial shear strength is increased to 62.4 kPa and the overall tear resistance reaches 1.42 MPa. Due to the high extracellular matrix content of bovine muscle tissue and more thorough freeze-hardening treatment, the inner wall structure of the pores is more compact, forming a stronger mechanical interlocking effect with the expanded artificial blood vessel.

[0105] Example 3 uses axial tensile shrinkage instead of temperature treatment. The interfacial shear strength is 55.8 kPa and the overall tear strength is 1.21 MPa, which is similar to Example 1. This shows that both shrinkage-expansion mechanisms can achieve reliable interfacial bonding. The axial tensile method is simpler to operate and is especially suitable for temperature-sensitive material systems.

[0106] Example 4 increased the number of openings and increased the interference fit (the difference between the outer diameter and the inner diameter is 0.8 mm). The interfacial shear strength was 60.1 kPa and the overall tear strength was 1.38 MPa, indicating that appropriately increasing the interference fit helps to improve the interfacial bonding strength.

[0107] Example 5 uses a small-diameter polycaprolactone artificial blood vessel. Due to the size effect and the relatively soft nature of the material itself, the interfacial shear strength is 47.5 kPa and the overall tear strength is 0.95 MPa. Although these values ​​are lower than those of other examples, they are still superior to the comparative examples and meet the application requirements of microvascular flaps.

[0108] Comparative Example 1 did not undergo cryo-hardening treatment and directly made a hole in the soft muscle tissue, which resulted in tissue collapse and irregular structure at the edge of the hole. After the artificial blood vessel was implanted, the contact area was reduced and the fit was loose. The interfacial shear strength was only 29.8 kPa and the overall tear resistance was 0.62 MPa.

[0109] Comparative Example 2 omitted the expansion process, and the artificial blood vessel and the opening were in a gap fit, lacking radial clamping force. The interfacial shear strength dropped sharply to 7.5 kPa, and the overall tear resistance was 0.35 MPa. This indicates that the radial compressive stress generated by the interference fit is the core reason for the formation of effective interfacial bonding. In clinical applications, flaps without expansion are very prone to displacement or detachment of the artificial blood vessel.

[0110] In summary, the vascularized flap prepared by the method of the present invention can achieve tight mechanical integration, has high interfacial shear strength and overall tear resistance, which is beneficial to the practical application of vascularized flaps.

[0111] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for preparing a vascularized flap, characterized in that, include: Provide muscle tissue and artificial blood vessels; The muscle tissue is cooled, and the cooled muscle tissue is then perforated to obtain the first structure. The artificial blood vessel is contracted, and the contracted artificial blood vessel is implanted into the opening of the first structure to obtain the second structure. The artificial blood vessel in the second structure is expanded to obtain the third structure; The third structure is decellularized to obtain a vascularized flap; wherein, The outer diameter of the artificial blood vessel is larger than the inner diameter of the opening.

2. The method for preparing a vascularized flap according to claim 1, characterized in that, The cooling treatment temperature is -20℃ to 4℃; and / or, The cooling process takes 2-24 hours.

3. The method for preparing a vascularized flap according to claim 1, characterized in that, The shrinkage process includes a cooling process, and the expansion process includes a heating process.

4. The method for preparing a vascularized flap according to claim 3, characterized in that, The cooling treatment temperature is 0℃-5℃; and / or, The cooling process takes 1-4 hours; and / or, The temperature for the heating treatment is 25℃-37℃; and / or, The heating process takes 1-6 hours.

5. The method for preparing a vascularized flap according to claim 1 or 3, characterized in that, The shrinkage treatment includes axial stretching treatment, and the expansion treatment includes releasing the axial stretching; and / or, The shrinkage process includes negative pressure compression, and the expansion process includes releasing the negative pressure compression.

6. The method for preparing a vascularized flap according to claim 1, characterized in that, The porosity of the vascularized flap is 65%-90%; and / or, In the aforementioned opening process, multiple openings are provided, with the center-to-center distance between adjacent openings being 2cm-5cm; and / or, In the opening process, multiple openings are provided, and the extension directions of the multiple openings are arranged in parallel; and / or, The decellularization process includes physical shaking and chemical washing.

7. The method for preparing a vascularized flap according to claim 1, characterized in that, The outer diameter of the artificial blood vessel is 0.2mm-1.5mm larger than the inner diameter of the opening; and / or, The inner diameter of the artificial blood vessel is 0.5mm-3mm; and / or, The artificial blood vessel comprises a biodegradable artificial blood vessel made of at least one biodegradable material selected from polyglycolic acid, polylactic acid-glycolic acid copolymer, polylactic acid, polydioxanone, or polycaprolactone; and / or, The inner wall of the artificial blood vessel is provided with a bioactive coating, the bioactive coating comprising at least one of gelatin, chitosan, collagen, or vascular endothelial growth factor; and / or, The muscle tissue includes at least one of the muscle tissues of pigs, cattle, sheep, and rabbits.

8. The method for preparing a vascularized flap according to claim 1, characterized in that, The artificial blood vessel includes an interconnected connecting segment and an anastomotic segment, the connecting segment being disposed within the opening and the anastomotic segment extending to the outside of the muscle tissue.

9. The method for preparing a vascularized flap according to claim 8, characterized in that, The thickness of the muscle tissue is 3mm-15mm; and / or, The length of the anastomotic segment is 2mm-10mm; and / or, The length of the artificial blood vessel is greater than the length of the opening, so as to form two anastomotic segments outside the muscle tissue.

10. A vascularized flap, characterized in that, It is prepared by the method for preparing vascularized flaps according to any one of claims 1 to 9.

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